Organ transplant therapy is increasingly used for the clinical treatment of diseased organs. Expanded use of this treatment will come only when mechanisms for the long term storage of donor organs are found. Cryopreservation is an obvious answer yet, to date, freezing of whole organs has been largely unsuccessful; the technology that permits cryopreservation of isolated cells fails when faced with the complexities of the whole organ. The problems and solutions to organ cryopreservation can be attacked directly through studies of vertebrate model animals that show natural abilities to endure extended bouts of freezing; our model animals, wood frogs Rana sylvatica and painted turtle hatchlings Chrysemys picta, readily survive days or weeks of freezing during natural hibernation with up to 65 % of their total body water as extracellular ice. NIH-funded studies in my lab have, to date, analyzed critical areas of natural freeze tolerance including an organ-specific analysis of the stresses of freezing, the actions of cryoprotectants, and metabolic responses to the frozen state. Of key importance, we have demonstrated that freezing survival entails not only measures for the physical protection of cells (e.g. cryoprotectant synthesis, ice nucleating proteins) but also numerous pro-active metabolic responses that are initiated to prepare cells and organs to endure the ischemia and cell volume changes that result from the freezing of extracellular fluids. Proposed studies will provide a comprehensive analysis of the pro-active metabolic responses to freezing by organs of freeze tolerant frogs and turtles including changes in protein biosynthesis, the expression of stress-related proteins, the roles of adrenoceptors and of protein kinases A and C in mediating cellular responses, the role of cell volume changes in stimulating the expression of the metabolic responses involved in freezing survival, the involvement of anti-oxidant systems in freezing protection, the effects of cold and freezing on mitochondrial metabolism, and the characterization of vertebrate ice nucleating proteins. Parallel studies will provide a qualitative and quantitative evaluation of the metabolic responses to freezing by mammalian cells (hepatocytes) that will allow us to identify those responses that are positive and adaptive for freezing survival versus those that are maladaptive. From this information, we will then design and implement intervention strategies for mammalian cells and organs that will allow us to manipulate the metabolism of mammalian cells to improve their viability during long term freezing storage.